Expression and secretion vector for human interferon alpha and process for producing human interferon alpha by employing same

ABSTRACT

Disclosed in this invention are an expression vector for the secretive production of human interferon alpha (hIFNα) comprising a polynucleotide encoding a modified  E. coli  thermostable enterotoxin II signal sequence and a polynucleotide encoding hIFNα ligated to the 3′-end thereof; a microorganism transformed with the expression vector; and a process for secretively producing human interferon by culturing the microorganism, the process being capable of secreting a soluble form of active hIFNα, which does not contain an additional methionine residue at its N-terminal, into the periplasm of an  E. coli  cell.

FIELD OF THE INVENTION

The present invention relates to an expression vector for the secretiveproduction of human interferon alpha (hIFNα) comprising a polynucleotideencoding a modified E. coli thermostable enterotoxin II signal sequenceand a polynucleotide encoding hIFNα ligated to the 3′-end thereof; amicroorganism transformed with the expression vector; and a process forsecretively producing hIFNα having no methionine residue added at itsN-terminal in the periplasm of E. coli cell.

BACKGROUND OF THE INVENTION

Isaacs and Lindenmann reported in 1957 that when chicken is infectedwith influenza virus A, a viral replication inhibitory factor designatedinterferon is produced (Isaacs, K and Lindenmann, J. Proc. R. Soc.Lond., B147:258–267, 1957).

Human interferons are cytokine proteins which inhibit in vivo immuneresponse or viral replication and they are classified as interferonalpha (IFNα), interferon beta (IFNβ) and interferon gamma (IFNγ)according to cell types producing them (Kirchner, H. et al., Tex. Rep.Biol. Med., 41:89–93, 1981; Stanton, G. J. et al., Tex. Rep. Biol. Med.,41:84–88, 1981).

It is well-known that these interferons work together to exert synergiceffects in the manifestation of anti-viral, anti-cancer, NK (naturalkiller) cell activation and marrow cell inhibition activities (Klimpel,et al. J. Immunol., 129:76–78, 1982; Fleischmann, W. R. et al., J. Natl.Cancer Inst., 65:863–966, 1980; Weigent, et al., Infec. Immun.,40:35–38, 1980). In addition, interferons act as regulatory factors ofthe expression, structure and function of genes in the cell, and show adirect anti-proliferating effect.

IFNα is produced when leukocyte is stimulated by B cell mitogen, virusor cancer cells. Up to now, there have been reported genes that encodemore than 20 species of interferons, each comprising 165 or 166 aminoacids.

IFNα used for early clinical tests were obtained from buffy coatleukocyte stimulated by Sendai virus and its purity was only less than1% (Cantell, K. and Hirvonen, Tex. Rep. Biol. Med., 35:138–144, 1977).

It has become possible to produce a large quantity of IFNα havingbiophysical activity by gene recombinant techniques in the 1980'(Goedell, D. V. et al., Nature, 287:411–416, 1980). Clinical tests usingthe recombinant hIFNα have shown that it is effective in treatingvarious solid cancers, particularly bladder cancer, kidney cancer, HIVrelated Kaposi's sarcoma, etc. (Torti, F. M., J. Clin. Oncol.,6:476–483, 1988; Vugrin, D., et al., Cancer Treat. Rep., 69:817–820,1985; Rios, A., et al., J. Clin. Oncol., 3:506–512, 1985). It is alsoeffective for the treatment of hepatitis C virus (Davis, G. G., et al.,N. Engl. J. Med., 321:1501–1506, 1989), and its applicable range as atherapeutic agent is expanding day by day.

The result of cloning IFNα gene from leukocyte has shown that IFNα isencoded by a group of at least 10 different genes. This indicates thatthe DNA sequences of the genes do not produce one kind of protein butthat IFNα is a mixture of subtype proteins having similar structures.Such subtype proteins are named IFNα-1, 2, 3, and so on (Nature,290:20–26, 1981).

Among the several types of interferons, hIFNα purified from humanleukocyte has a molecular weight of 17,500 to 21,000, and a very highnative activity of about 2×10⁸ IU/mg protein. In vivo IFNα is a proteinconsisting of 165 amino acids. It is designated IFNα-2a (SEQ ID NO: 1)in case the 23^(rd) amino acid is lycine, and IFNα-2b (SEQ ID NO : 2) incase the 23^(rd) amino acid is arginine. In the beginning hIFNα wasproduced by a process using a cell culture method. However, this processis unsuitable for commercialization because of its low productivity ofabout 250 ug/L.

To solve this problem, processes for recovering a large quantity ofinterferon from microorganisms by using gene recombinant techniques havebeen developed and used to date.

The most widely employed is a process using E. coli which produces IFNαconsisting of 166 or 167 amino acids according to the characteristics ofthe E. coli cell. These products have an extra methionine residue addedat the N-terminal by the action of the ATG codon existing at the site ofinitiation codon. However, it has been reported that the additionalmethionine residue can trigger harmful immune response, in the case ofhuman growth hormone (EP Patent Publication No. 256,843).

In addition, most of the expressed IFNα accumulates in cytoplasm in theform of insoluble inclusion bodies and must be converted into an activeform through refolding during a purification process. As such arefolding process is not efficient, IFNα exists partially in a reducedform, or forms an intermolecular disulfide coupling body or a defectivedisulfide coupling body. It is difficult to remove these by-products,which cause a markedly low yield. In particular, it is extremelydifficult to remove undesirable interferon by-products such as misfoldedinterferons.

Recently, in order to solve the above mentioned problems associated withthe production of a foreign protein within a microbial cell, variousefforts have been made to develop a method based on efficient secretionof a soluble form of the target protein carrying no extra methionineadded to the N-terminal.

In this method, a desired protein is expressed in the form of a fusionprotein which carries a signal peptide attached to its N-terminal. Whenthe fusion protein passes through the cell membrane, the signal peptideis removed by an enzyme in E. coli and the desired protein is secretedin a native form.

The secretive production method is more advantageous than the microbialproduction method in that the amino acid sequence and the higherstructure of the produced protein are usually identical to those of thewild-type. However, the yield of a secretive production method is oftenquite low due to its unsatisfactory efficiencies in both the membranetransport and the subsequent purification process. This is in line withthe well-known fact that the yield of a mammalian protein produced in asecretory mode in prokaryotes is much lower than that of a prokaryoticprotein produced in the same mode in prokaryotes. Therefore, it has beenattempted to develop a more efficient secretory production method. Forinstance, Korean Patent Publication No. 93-1387 discloses an attempt tomass-produce IFNα using the signal peptide of E. coli alkalinephosphatase, but the yield was very low at 10⁹ IU/L culture medium (10ug/L culture medium). Therefore, there has been a keen interest indeveloping a method which is capable of producing soluble IFNα having noadditional methionine residue added at the N-terminal, using amicroorganism on a large scale.

The present inventors have previously generated a new signal peptide ofE. coli thermostable enterotoxin II (Korean Patent Application No.98-38061 and 99-27418) and found that this new secretory signal peptidecan be used for the mass-production of the native form of IFNα. Namely,the present inventors have constructed an expression vector containing agene obtained by ligating IFNα encoding gene instead of enterotoxin IIencoding gene to the modified E. coli secretory signal peptide, anddeveloped a secretory production method of IFNα having a nativebiological activity via the microbial secretory system by culturing themicroorganism transformed with said expression vector.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide anexpression vector which can secretively produce human interferon alpha(hIFNα).

It is another object of the present invention to provide a microorganismtransformed with said expression vector.

It is a further object of the present invention to provide a process forproducing a soluble form of hIFNα using said microorganism, which has noextra methionine residue attached to the amino terminus.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention taken inconjunction with the following accompanying drawings; which respectivelyshow:

FIG. 1: the procedure for constructing vector pT-IFNα-2a;

FIG. 2: the procedure for constructing vector pT14SIα-2a;

FIG. 3: the procedure for constructing vector pT14SSIα-2a;

FIG. 4: the procedure for constructing vector pT140SSIα-2a-4T22Q;

FIGS. 5 a and 5 b: the results of SDS-PAGE which verify the expressionof IFNα-2a and the purity of the expressed IFNα-2a from recombinant celllines, and the result of western blot analysis which verifies themolecular weight of expressed IFNα-2b, respectively.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided anexpression vector for the secretive production of hIFNα comprising apolynucleotide encoding a modified thermostable enterotoxin II signalsequence (hereinafter, as referred as to ‘STII mutant’) and apolynucleotide encoding hIFNα ligated to the 3′-end thereof.

The polynucleotide encoding hIFNα used for constructing the expressionvector of the present invention may be any one of polynucleotidesencoding random hIFNα subtypes such as native hIFNα-2a (SEQ ID NO: 1),IFNα-2b (SEQ ID NO : 2), IFNα-1 and IFNα-3, and it may also be arecombinant polynucleotide which has a modified base sequence thatencodes any of the above IFNα subtypes.

The polynucleotide encoding the modified E. coli thermostableenterotoxin II signal sequence of the present invention, which isligated to the front of the 5′-end of the polynucleotide encoding hIFNαand used for the purpose of the secretive production of hIFNα, may be apolynucleotide encoding a mutant derivable by replacing one or more ofthe amino acids of E. coli thermostable enterotoxin II signal sequencedescribed in SEQ ID NO: 3, preferably one or more of the 4^(th), 20^(th)and 22^(nd) amino acids thereof with other amino acid(s). Examples ofsuch polynucleotides encode mutants obtained by replacing: the 4^(th)amino acid with threonine ([Thr⁴]STII); the 4^(th) amino acid withthreonine and the 22^(nd) amino acid with glutamine, respectively([Thr⁴, Gln²²]STII); the 4^(th) amino acid with threonine, the 20^(th)amino acid with valine and the 22^(nd) amino acid with glutamine,respectively ([Thr⁴, Val²⁰, Gln²²]STII); and the 4^(th) amino acid withthreonine and the 20^(th) amino acid with valine, respectively ([Thr⁴,Val²⁰]SDII) in the E. coli thermostable enterotoxin II signal sequence(STII) described in the SEQ ID NO: 3, and preferred polynucleotidesequences are SEQ ID NOS: 4, 5, 6 and 7. However, it is known thatseveral different polynucleotides encoding the mutants of the presentinvention may exist due to the codon degeneracy, and, specifically, apolynucleotide modified by introducing preferred codons of E. coliwithout any change of amino acid sequence can be used for promoting theexpression rate of IFNα.

In addition, the expression vector of the present invention may furthercomprise E. coli thermostable enterotoxin II Shine-Dalgarno sequence (SDsequence, SEQ ID NO: 8) or its mutant ligated to the front of the 5′-endof the polynucleotide encoding the modified thermostable enterotoxin IIsignal sequence. As compared with an wild-type which has 7 bases(TGATTTT) following GAGG of the 5′-end in the E. coli thermostableenterotoxin II SD sequence described in the SEQ ID NO: 8, the mutant ofSD sequence has a shorter sequence of 6 or 5 bases. The use of thismutant can increase the secretive expression rate of IFNα. However, whensaid base sequence becomes shorter than 4 bases, the expression ratedecreases markedly. A specific example of a preferred mutant that can beused in the present invention is the E. coli thermostable enterotoxin IISD sequence mutant having the nucleotide sequence of SEQ ID NO: 9.

The promoter used in preparing the expression vector of the presentinvention may be any of those which can express a heterologous proteinin a microorganism host. Specifically, lac, Tac, and arabinose promoteris preferred when the heterologous protein is expressed in E. coli.

This invention also provides transformed microorganisms which may beobtained e.g., by transforming such E. coli strains as E. coli BL21(DE3)(Novagen, USA) or E. coli XL-1 blue (Novagen, USA) with said expressionvector. Examples of the present invention provide such transformedmicroorganisms: E. coli BL21(DE3)/pT140SSIα-2a-4T (“HM 10603”), E. coliBL21(DE3)/pT140SSIα-2a-4T22Q (“UM 10611”), E. coliBL21(DE3)/pT140SSIα-2b-4T (“HM 10703”) and E. coliBL21(DE3)/pT140SSIα-2b-4T22Q (“HM 10711”). The above transformedmicroorganisms are deposited in Korean Culture Center of Microorganisms(KCCM) (Address; Yurim Bldg., 361-221, Hongje 1-dong, Seodaemun-gu,Seoul 120-091, Republic of Korea) on Dec. 23, 1999 under accessionnumbers KCCM-10175, KCCM-10176, KCCM-10177 and KCCM-10178, respectively,in accordance with the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

In accordance with another aspect of this invention, there is alsoprovided a process for secretively producing hIFNα having no additionalmethionine residue attached at the N-terminal, into the periplasm of E.coli by culturing the transformed microorganism under an appropriateculture condition which may be the same as the conventional culturecondition used for transformed microorganisms.

hIFNα secretively produced by the process of the present inventioncomprises random hIFNα subtypes such as IFNα-1, IFNα-3 and so on, aswell as native hIFNα-2a (SEQ ID NO: 1) and hIFNα-2b (SEQ ID NO: 2)consisting of 165 amino acids. In addition, the process of the presentinvention can be applied to the production of any other interferon suchas hIFNβ and hIFNγ.

According to the process of the present invention, 80% or more of IFNαproduced by the inventive E. coli transformant is secreted into theperiplasm at a high productivity of more than 1 g/L. The produced IFNαhas the same amino acid sequence as that of native IFNα which has noadditional amino acid attached at the N-terminal, and shows a biologicalactivity equal to that of native IFNα.

The following Examples are included to further illustrate the presentinvention without limiting its scope.

REFERENCE EXAMPLE IFNα-2a Gene and Construction of a Vector ContainingSame

A gene encoding hIFNα-2a was prepared by carrying out PCR using humangenomic DNA as a template and SEQ ID NOS: 10 and 11 as primers. Theprimer of SEQ ID NO: 10 was designed to provide an NdeI restriction site(5′-CATATG-3′) upstream from the codon for the first amino acid(cysteine) codon of native hIFNα, and the primer of SEQ ID NO: 11, toprovide a BamHI restriction site (5′-GGATCC-3′) downstream from thetermination codon thereof.

The amplified PCR product was cleaved with NdeI and BamHI to obtain aDNA fragment encoding hIFNα-2a. The DNA fragment was inserted into theNdeI/BamHI site of vector pET-14b (Novagen, USA) to obtain vectorpT-IFNα-2a.

FIG. 1 shows the above procedure for constructing vector pT-IFNα-2a.

COMPARATIVE EXAMPLE 1 Construction of a Vector Containing EnterotoxinSignal Sequence and IFNα-2a Genes

To prepare E. coli enterotoxin II signal sequence gene, the pair ofcomplementary oligonucleotides of SEQ ID NOS: 12 and 13 were designedbased on the previously known nucleotide sequence of E. coli enterotoxinII signal peptide, and synthesized using a DNA synthesizer (Model 380B,Applied Biosystem, USA). The above oligonucleotides were designed toprovide a BspHI restriction site (complementary sites to an NdeIrestriction site) upstream from the initiation codon of E. colienterotoxin II and a MluI restriction site introduced by a silent changeat the other end. Both oligonucleotides were annealed at 95° C. toobtain a blunt-ended DNA fragment having a nucleotide sequence encodingE. coli enterotoxin II signal sequence. The above DNA fragment wasinserted into the SmaI site of vector pUC19 (BioLabs, USA) to obtainvector pUC19ST.

In addition, vector pT-IFNα-2a containing IFNα-2a gene obtained inReference Example was subjected to PCR using the primers of SEQ ID NOS:14 and 15 to ligate the enterotoxin signal peptide to IFNα-2a gene. Theprimer of SEQ ID NO: 14 was designed to correspond to the 5′-end ofIFNα-2a gene, and the primer of SEQ ID NO: 15, to provide a BamHIrestriction site (5′-GGATCC-3′) downstream from the termination codonthereof. The DNA fragment containing the polynucleotide, which encodesnative IFNα-2a, was amplified by PCR using the above polynucleotideprimers. The amplified DNA fragment was cleaved with MluI and BamHI toobtain an IFNα-2a DNA fragment having MluI/BamHI ends.

Meanwhile, vector pUC19ST containing the enterotoxin signal peptide wascleaved with MluI and then digested with BamHI to obtain a vectorfragment having MluI/BamHI ends. The vector fragment was ligated to theIFNα-2a DNA fragment to construct vector pUC19SIFNα-2a.

Vector pUC19SIFNα-2a was cleaved with BspHI and BamHI to obtain a DNAfragment (564 bp). The DNA fragment was inserted at the NcoI/BamHIsection of vector pET-14b (Novagen, USA) to obtain vector pT14SIα-2a.FIG. 2 shows the above procedure for constructing vector pT14SIα-2a.

Subsequently, E. coli BL21(DE3) strain was treated with 70 mM calciumchloride solution to prepare competent E. coli, and then, vectorpT141α-2a in 10 mM Tris buffer (pH 7.5) was added thereto. An E. colitransformant expressing IFNα-2a was selected by a conventional methodwhich exploits the sensitivity of the transformed vector towardantibiotics, and designated E. coli HM 1 0600.

In addition, vector pT14SIα-2a was subjected to PCR using the primers ofSEQ ID NOS: 16 and 17 to amplify a DNA fragment obtained by ligating theShine-Dalgarno sequence of the enterotoxin, the enterotoxin signalpeptide, and IFNα-2a gene, in that order, and then the DNA fragment wascleaved with XbaI and BamHI to obtain an insert.

The insert was ligated into the XbaI/BamHI section of vector pET-14b(Novagen, USA) to construct vector pT14SSIα-2a. FIG. 3 displays theabove procedure for constructing vector pT13SSIα-2a. E. coli BL21(DE3)(Stratagene, USA) was transformed with vector pT14SSIα-2a to obtain atransformant designated E. coli HM 10601.

COMPARATIVE EXAMPLE 2 Construction of a Vector Containing EnterotoxinSignal Sequence and IFNα-2b Genes

The 23^(rd) lycine codon of IFNα-2a gene in vector pT14SSIα-2a wasreplaced by arginine codon with a site-directed mutagenesis (Papworth,C. et al., Stratagies, 9, 3, 1996) to construct an expression vectorcontaining IFNα-2b gene. Vector pT14SSIα-2a was subjected tohybridization with the synthetic oligonucleotides of SEQ ID NOS: 19 and20 containing the replaced codon to form a hybrid molecule and DNAamplification was performed using pfu (Stratagene, USA) and fournucleotide triphosphates (ATP, GTP, TTT, CTP) which extend saidoligonucleotides in the 5′-3′ direction.

Interferon α-2b sequence17  18  19  20  21  22  23  24  25  26  27  28  29 Leu Leu Ala Gln MetArg Arg Ile Ser Leu Phe Ser Cys (SEQ ID NO:18) CTC CTG GCA CAG ATG AGGAGA ATC TCT CTT TTC TCC TGC (SEQ ID NO:19) GCA GGA AGG AAG AGA GAT TCTCCT CAT CTG TGC CAG GAG (SEQ ID NO:20)

The amplified DNA fragment was recovered and an restriction enzyme DpnIwas added thereto to remove unconverted plasmids completely.

E. coli XL-1 blue (Novagen, USA) was transformed with the modifiedplasmid. The base sequence of the DNA recovered from transformedcolonies was determined, and thus obtained was plasmid pT14SSIα-2b whichcontained a gene having arginine in place of the 23^(rd) amino acidlycine of IFNα2a.

Subsequently, E. coli BL21(DE3) was transformed with the modified vectorpT14SSIα-2b to obtain a transformant designated E. coli HM10701 by usingthe same method described in Comparative Example 1. By analyzing theN-terminal amino acid sequence of the protein produced by culturing thetransformant, it has been confirmed that IFNα-2b having the native aminoacid sequence was expressed therefrom.

EXAMPLE 1 Construction of a Vector Containing Enterotoxin Signal PeptideMutant

(1) Construction of a Vector Containing [Thr⁴]STII

In order to modify a specific amino acid residue of the enterotoxinsignal sequence peptide, a vector containing a polynucleotide encodingenterotoxin mutant signal sequence was prepared by site-directedmutagenesis as follows.

First, vector pT14SSIα-2a obtained in Comparative Example 1 wassubjected to PCR using oligonucleotides of SEQ ID NOS: 22 and 23 toobtain a modified plasmid, wherein the 4^(th) amino acid of theenterotoxin signal sequence is replaced with threonine (Thr), by thesite-directed mutagenesis procedure described in Comparative Example 2.

(SEQ ID NO:21)              Met Lys Lys Thr Ile Ala Phe Leu (SEQ IDNO:22) 5′-GGTGATTTT ATG AAA AAG ACA ATC GCA TTT CTT C-3′ (SEQ ID NO:23)3′-CCACTAAAA TAC TTT TTC TGT TAG CGT AAA GAA G-5′

Then, E. coli XL-1 blue (Novagen, USA) was transformed with the modifiedplasmid. The base sequence of DNA recovered from the transformedcolonies was determined, and thus obtained was a plasmid which containeda gene encoding the enterotoxin signal sequence peptide having Thr inthe 4^(th) amino acid position thereof. The plasmid thus obtained wascleaved with XbaI and MluI, and then inserted into the XbaI/MluI sectionof vector pT14SSIα-2a to obtain vector pT14SSIα-2a-4T.

Subsequently, E. coli BL21(DE3) (Stratagene, USA) was transformed withvector pT14SSIα-2a-4T to obtain an E. coli transformant designated E.coli HM 10602.

Vector pT14SSIα-2a-4T was constructed using pT14SSIα-2b, and thentransformed into E. coli BL21(DE3) (Stratagene, USA) to obtain an E.coli transformant designated E. coli HM 10702 by the same methoddescribed above.

(2) Construction of a Vector Containing [Thr⁴, Gln²²]STII

Vector pT14SSIα-2a-4T obtained in step (1) was subjected to PCR usingthe oligonucleotides of SEQ ID NOS: 25 and 26, which were designed tosubstitute Gln codon for the 22^(nd) amino acid of the enterotoxinsignal peptide having Thr in its 4^(th) position, in accordance with thesite-directed mutagenesis procedure of step (1) to obtain a modifiedplasmid.

(SEQ ID NO:24)       Asp Ala Gln Ala Cys Asp Leu Pro (SEQ ID NO:25)5′-CA ATT GCC CAA GCG TGT GAT CTG CCT-3′ (SEQ ID NO:26) 3′-GT TTA CGGGTT CGC ACA CTA GAC GGA-5′

Then, E. coli XL-1 blue (Novagen, USA) was transformed with the modifiedplasmid. The base sequence of DNA recovered from transformed colonieswas determined, and thus obtained was plasmid pT14SSIα-2a-4T22Q whichcontained a gene having Thr and Gln in the 4^(th) and 22^(nd) amino acidpositions of the enterotoxin signal sequence, respectively.Subsequently, E. coli BL21(DE3) (Stratagen, USA) was transformed withvector pT14SSIα-2a-4T22Q by the same method described in step (1) toobtain a transformant designated E. coli HM 10604.

To modify the Shine-Dalgarno sequence of the modified enterotoxin signalsequence into SEQ ID NO: 9, vectors pT14SSIα-2a-4T and pT14SSIα-2a-4T22Qwere subjected to the site-directed mutagenesis procedure described instep (2) using the oligonucleotides of SEQ ID NOS: 27 and 28 to obtainthe desired modified plasmid.

E. coli XL-1 blue (Novagen, USA) was transformed with the modifiedplasmid. The base sequence of the DNA recovered from transformedcolonies was determined, and thus obtained were plasmids pT14OSSIα-2a-4Tand pT14OSSIα-2a-4T22Q having modified Shine-Dalgarno sequence ofenterotoxin signal sequence. FIG. 4 represents the above procedure forconstructing vector pT14OSSIα-2a-4T22Q.

E. coli BL21(DE3) was transformed with vector pT14OSSIα-2a-4T andpT14OSSIα-2a-4T22Q, respectively, to obtain a transformant designated E.coli HM 10603 and HM 10611, which were deposited in Korean CultureCollection of Microorganisms (KCCM) on Dec. 23, 1999 under accessionnumbers KCCM-10175 and KCCM-10176, respectively.

In addition, vectors pT14OSSIα-2b-4T and pT14OSSIα-2b-4T22Q wereprepared by the same procedure as above using vector pT14SSIα-2b, whichwere used to transform E. coli BL21(DE3) to obtain transformantsdesignated E. coli HM 10703 and HM 10711, respectively. E. colitransformants HM 10703 and HM 10711 were deposited in KCCM on Dec. 23,1999 under accession numbers KCCM-10177 and KCCM-10178, respectively.

(3) Construction of a Vector Containing [Thr⁴, Val²⁰, Gln²²]STII

To further substitute Val codon for the 20^(th) amino acid of theenterotoxin signal sequence peptide having Thr and Gln in its 4^(th) and22^(nd) amino acid positions, vectors pT14OSSIα-2a-4T22Q andpT14OSSIα-2b-4T22Q prepared in step (2) were subjected to PCR using theoligonucleotides of SEQ ID NOS: 29 and 30 by the site-directedmutagenesis procedure described in step (2), to obtain the desiredmodified plasmids designated pT14OSSIα-2a-4T20V22Q andpT14OSSIα-2b-4T20V22Q.

E. coli XL-1 blue was transformed with the modified plasmids. The basesequences of the DNAs recovered from transformed colonies weredetermined, and thus obtained were plasmids pT14OSSIα-2a-4T20V22Q andpT14OSSIα-2b-4T20V22Q which contained a gene having Thr, Val and Glncodons in places of the 4^(th) Asp, 20^(th) Asp and 22^(nd) Tyr codons,respectively. E. coli BL21(DE3) was transformed with the plasmids toobtain thransformants designated E. coli HM 10612 and HM 10712,respectively.

EXAMPLE 2 Preparation of Thermostable Enterotoxin II Shine-DalgarnoSequence Mutant

In order to reduce the number of bases between the ribosome binding siteand initiation codon ATG of the modified E. coli thermostableenterotoxin II signal sequence within thermostable enterotoxin IIShine-Dalgarno sequence of the above-prepared expression vector, amodified plasmid was constructed by the site-directed mutagenesisprocedure of Comparative Example 2.

Namely, to reduce the number of bases between the ribosome binding siteGAGG and initiation codon ATG from 7 to 5, vector pT14OSSIα-2a-4T22Qprepared in Example 1 (2) was subjected to the site-directed mutagenesisprocedure of Comparative Example 2 using the oligonucleotides of SEQ IDNOS: 31 and 32 to obtain a modified plasmid designatedpT14NSSIα-2a-4T22Q. In addition, to reduce the number of bases betweenthe ribosome binding site GAGG and initiation codon ATG to 4, vectorpT14NSSIα-2a-4T22Q was subjected to by the site-directed mutagenesisprocedure of Comparative Example 2 using the oligonucleotides of SEQ IDNOS: 33 and 34 to obtain a modified plasmid designatedpT14MSSIα-2a-4T22Q.

E. coli XL-1 blue was transformed with the modified plasmids. The basesequences of the DNAs recovered from transformed colonies weredetermined, and thus obtained were IFNα expression plasmidspT14NSSIα-2a-4T22Q and pT14MSSIα-2a-4T22Q which respectively contained 5and 4 bases between the ribosome binding site GAGG and initiation codonATG. E. coli BL21(DE3) was transformed with the expression plasmids toobtain transformants designated HM 10613 and HM 10614, respectively.

EXAMPLE 3 Comparison of Expression Amount of IFNα-2

Transformants prepared in the above Comparative Examples and Exampleswere cultured in LB medium and then incubated in the presence of IPTGfor 3 hours. Each of the cultures was centrifuged at 6,000 rpm for 20min. to precipitate bacterial cells and the precipitate was treated byOsmotic shock method (Nossal, G. N., J. Biol. Chem., 241:3055, 1966) asfollowing.

The precipitate was suspended in a 1/10 volume of isotonic solution (20%sucrose, 10 mM Tris-Cl buffer containing 1 mM EDTA, pH 7.0). Thesuspension was allowed to stand at room temperature for 30 min, and thencentrifuged to collect bacterial cells. The cells were resuspended inD.W. at 4° C. to extract the proteins present in the periplasm of thecells, and centrifuged to obtain a supernatant as a periplasmicsolution. The IFNα-2 level in the periplasmic solution was assayed inaccordance with ELISA method (Kato, K. et al., J. Immunol., 116, 1554,1976) using an antibody against the IFNα-2 (R&D, USA), which wascalculated as the amount of the IFNα-2a produced per 1 l of culture. Theresults are shown in Table 1.

TABLE I Comparision of expression amount of IFNα-2 Modified IFNα-2Expression amino acid Level Transformant Example Vector residue in STIIin periplasm* HM 10600 Comp. PT14SIα-2a 82 ± 40 Exam. 1 HM 10601 Comp.PT14SSIα-2a 325 ± 75  Exam. 1 HM 10701 Comp. PT14SSIα-2b 288 ± 90  Exam.2 HM 10602 Example 1 (1) pT14SSIα-2a-4T Thr⁴ 550 ± 120 HM 10603 Example1 (2) pT14OSSIα-2a-4T Thr⁴ 1,020 ± 135   HM 10604 Example 1 (2)PT14SSIα-2a-4T22Q Thr⁴, Gln²² 680 ± 105 HM 10611 Example 1 (2)pT14OSSIα-2a-4T22Q Thr⁴, Gln²² 1,220 ± 120   HM 10612 Example 1 (3)pT14OSSIα-2a-4T20V22Q Thr⁴, Val²⁰, Gln²² 1,130 ± 180   HM 10613 Example2 pT14NSSIα-2a-4T22Q Thr⁴, Gln²² 750 ± 144 HM 10614 Example 2pT14MSSIα-2a-4T22Q Thr⁴, Gln²² 420 ± 100 HM 10702 Example 1 (1)pT14SSIα-2b-4T Thr⁴ 370 ± 90  HM 10703 Example 1 (2) pT14OSSIα-2b-4TThr⁴ 735 ± 117 HM 10711 Example 1 (2) pT14OSSIα-2b-4T22Q Thr⁴, Gln²²1,070 ± 150   HM 10712 Example 1 (3) pT14OSSIα-2b-4T20V22Q Thr⁴, Val²⁰,Gln²² 820 ± 160 *IFNα mg/100 O.D_(600 nm)/L culture solution

EXAMPLE 4 Post-treatment and Purification

According to the procedure of Example 3, transformant E. coli HM 10611prepared in Example 1(2) was cultured in LB medium and the culture wascentrifuged for 6,000 rpm for 20 min. to harvest cells. The periplasmicsolution was prepared from the cells by the Osmotic Shock method.

The periplasmic solution was adjusted to pH 5.0 to 5.5, adsorbed on anS-Sepharose (Pharmacia Inc., Sweden) column pre-equilibrated to pH 5.3,and then, the column was washed with 25 mM NaCl. IFNα-2 was eluted bysequentially adding acetic acid buffer solutions containing 50 mM, 100mM, 200 mM and 1 M NaCl, respectively, and fractions containing IFNα-2were collected and combined.

The combined fractions were subjected to Blue Sepharose (Pharmacia Inc.,Sweden) column chromatography and eluted by adding to the column buffersolutions containing more than 2 M NaCl to obtain an active fraction.

The active fraction was dialyzed with a buffer, and finally subjected toresin column fractionation using a DEAE anion exchange resin column atpH 5.8 to obtain IFNα-2a having a purity of more than 99%. In addition,IFNα-2b was purified from transformant E. coli HM 10711 by repeating theabove procedure.

Each of the purified IFNα-2a and IFNα-2b fractions was subjected tosodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) todetermine the purity and approximate IFNα concentration, and thensubjected to a conventional ELISA method as in Example 3 to determinethe exact IFNα concentration in the periplasmic solution. In addition,it was confirmed by N-terminal amino acid sequence analysis that IFNα-2aand IFNα-2b were of the native types having no additional methionine.

EXAMPLE 5 Determination of IFNα-2a Molecular Weight Produced fromRecombinant Cell Lines

The expression and molecular weights of IFNα-2a and IFNα-2b producedfrom recombinant cell lines were determined by using SDS-PAGE andWestern blotting.

First, the periplasmic fraction of transformant E. coli HM 10611prepared in Example 4 and purified IFNα-2a obtained therefrom weresubjected to SDS-PAGE using a commercial IFNα-2a product (3×10⁶ IU/ml)as a control according to the conventional method. FIG. 5 a reproducesthe SDS-PAGE result, wherein lane 1 shows the IFNα-2a control; lane 2,the periplasmic fraction of E. coli transformant HM 10611; and lane 3,the purified IFNα-2a. As can be seen from FIG. 5 a, the purified IFNα-2ahad the same molecular weight as that of the native IFNα-2a, and waspresent in the periplasmic fraction of transformant E. coli HM 10611 ata high level.

In addition, the periplasmic fraction of transformant E. coli HM 10711,a purified fraction obtained by subjecting the periplasmic solution toS-Sepharose column chromatography and the finally purified IFNα-2b weresubjected to SDS-PAGE according to the conventional method.

A nitrocellulose filter (Bio-Rad Lab, USA) was wetted with a buffersolution for blotting (170 mM glycine, 25 mM Tris•HCl [pH 8], 20%methanol) and the proteins separated on the gel were transferred ontothe nitrocellulose filter over a period of 3 hours by using a blottingkit. The filter was kept in 1% Casein for 1 hour and washed three timeswith PBS containing 0.05% Tween 20. The filter was put in a rabbitanti-IFNα antibody (Chemicon, #AB1434, USA) solution diluted with PBSand reacted at room temperature for 2 hours. After reaction, the filterwas washed 3 times with a PBST solution to remove unreacted antibody.Horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Lab.,USA) diluted with PBS was added thereto and reacted at room temperaturefor 2 hour. The filter was washed with PBST, and a peroxidase substratekit (Bio-Rad Lab., USA) solution was added thereto to develop a colorreaction. The results from the above western blotting are shown in FIG.5 b, wherein lane 1 represents the periplasmic fraction of transformantE. coli HM 10711; lane 2, the fraction purified with S-Sepharose columnchromatography; and lane 3, the final purified IFNα-2b.

As a result of Example, it is confirmed that a large quantity of solubleIFNα is expressed from the recombinant E. coli strains of the presentinvention.

1. An expression vector for the secretive production of human interferonalpha (hIFNα) comprising a polynucleotide encoding a modifiedthermostable enterotoxin II signal sequence and a polynucleotideencoding hIFNα ligated to the 3′-end thereof, wherein the modifiedthermostable enterotoxin II signal sequence is selected from the groupconsisting of: a polypeptide obtained by replacing the 4^(th) asparagineof the amino acid sequence of SEQ ID NO: 3 with threonine; a polypeptideobtained by replacing the 4^(th) asparagine and 22^(nd) tyrosine of theamino acid sequence of SEQ ID NO: 3 with threonine and glutamine,respectively; a polypeptide obtained by replacing the 4^(th) and 20^(th)asparagines of the amino acid sequence of SEQ ID NO: 3 with threonineand valine, respectively, and; a polypeptide obtained by replacing the4^(th) asparagine, the 20^(th) asparagine and the 22^(nd) tyrosine ofthe amino acid sequence of SEQ ID NO: 3 with threonine, valine andglutamine, respectively.
 2. The expression vector according to claim 1,wherein the polynucleotide encoding hIFNα codes for IFNα-2a of SEQ IDNO: 1 or IFNα-2b of SEQ ID NO:2.
 3. The expression vector according toclaim 1, which further comprises E. coli thermostable enterotoxin IIShine-Dalgarno sequence (SD sequence, SEQ ID NO:8) or a mutant thereofligated to the front of the 5′-end of the polynucleotide encoding themodified thermostable enterotoxin II signal sequence, wherein the mutanthas the nucleotide sequence of SEQ ID NO:
 9. 4. The expression vectoraccording to claim 1, which is selected from the group consisting ofplasmids pT14SSIα-2a-4T, pT14OSSIα-2a-4T, pT14SSIα-2a-4T22Q,pT14OSSIα-2a-4T22Q, pT14OSSIα-2a-4T20V22Q, pT14NSSIα-2a-4T22Q,pT14MSSIα-2a-4T22Q, pT14SSIα-2b-4T, pT14OSSIα-2b-4T, pT14OSSIα-2b-4T22Qand pT14OSSIα-2b-4T20V22Q.
 5. An E. coli transformed with the expressionvector of any one of claims 1, 2, 3 and
 4. 6. The E. coli according toclaim 5, which is selected from the group consisting of E. coliBL21(DE3)/pT14SSIα-2a-4T (HM 10602), E. coli BL21(DE3)/pT14OSSIα-2a-4T(HM 10603; Accession NO: KCCM-10175), E. coliBL21(DE3)/pT14SSIα-2a-4T22Q (HM 10604), E. coliBL21(DE3)/pT14OSSIα-2a-4T22Q (HM 10611; Accession NO: KCCM-10176), E.coli BL21(DE3)/pT14OSSIα-2a-4T20V22Q (HM 10612), E. coliBL21(DE3)/pT14NSSIα-2a-4T22Q (HM 10613), E. coliBL21(DE3)/pT14MSSIα-2a-4T22Q (HM 10614), E. coliBL21(DE3)/pT14SSIα-2b-4T (HM 10702), E. coli BL21(DE3)/pT14OSSIα-2b-4T(HM 10703; Accession NO: KCCM-10177), E. coliBL21(DE3)/pT14OSSIα-2b-4T22Q (HM 10711; Accession NO: KCCM-10178) and E.coli BL21(DE3)/pT14OSSIα-2b-4T20V22Q (HM 10712).
 7. A process forsecretively producing hIFNα having no additional methionine residueattached at the N-terminal comprising the steps of transforming an E.coli with an expression vector for the secretive production of hIFNαcomprising a polynucleotide encoding a modified thermostable enterotoxinII signal sequence and a polynucleotide encoding hIFNα ligated to the3′-end thereof; and culturing the transformed E. coli, wherein themodified thermostable enterotoxin II signal sequence is selected fromthe group consisting of: a polypeptide obtained by replacing the 4^(th)asparagine of the amino acid sequence of SEQ ID NO: 3 with threonine; apolypeptide obtained by replacing the 4^(th) asparagine and 22^(nd)tyrosine of the amino acid sequence of SEQ ID NO: 3 with threonine andglutamine, respectively; a polypeptide obtained by replacing the 4^(th)and 20^(th) asparagines of the amino acid sequence of SEQ ID NO: 3 withthreonine and valine: and a polypeptide obtained by replacing the 4^(th)asparagine, the 20^(th) asparagine and the 22^(nd) tyrosine of the aminoacid sequence of SEQ ID NO: 3 with threonine, valine and glutamine,respectively.
 8. The process according to claim 7, wherein thepolynucleotide encoding hIFNα codes for IFNα-2a of SEQ ID NO: 1 orIFNα-2b of SEQ ID NO:
 2. 9. The process according to claim 7, which saidvector further comprises E. coli thermostable enterotoxin II SD sequence(SEQ ID NO:8) or a mutant thereof ligated to the front of the 5′-end ofthe polynucleotide encoding the modified thermostable enterotoxin IIsignal sequence, wherein the mutant has the nucleotide sequence of SEQID NO:
 9. 10. The process according to claim 7, wherein the expressionvector is selected from the group consisting of plasmids pT14SSIα-2a-4T,pT14OSSIα-2a-4T, pT14SSIα-2a-4T22Q, pT14OSSIα-2a-4T22Q,pT14OSSIα-2a-4T20V22Q, pT14NSSIα-2a-4T22Q, pT14MSSIα-2a-4T22Q,pT14SSIα-2b-4T, pT14OSSIα-2b-4T, pT14OSSIα-2b-4T22Q andpT14OSSIα-2b-4T20V22Q.
 11. The process according to claim 7, wherein thetransformed e. coli is selected from the group consisting of E. coliBL21(DE3)/pT14SSIα-2a-4T (HM 10602), E. coli BL21(DE3)/pT14OSSIα-2a-4T(HM 10603; Accession NO: KCCM-10175), E. coliBL21(DE3)/pT14SSIα-2a-4T22Q (HM 10604), E. coliBL21(DE3)/pT14OSSIα-2a-4T22Q (HM 10611; Accession NO: KCCM-10176), E.coli BL21(DE3)/pT14OSSIα-2a-4T20V22Q (HM 10612), E. coliBL21(DE3)/pT14NSSIα-2a-4T22Q (HM 10613), E. coliBL21(DE3)/pT14MSSIα-2a-4T22Q (HM 10614), E. coliBL21(DE3)/pT14SSIα-2b-4T (HM 10702), E. coli BL21(DE3)/pT14OSSIα-2b-4T(HM 10703; Accession NO: KCCM-10177), E. coliBL21(DE3)/pT14OSSIα-2b-4T22Q (HM 10711; Accession NO: KCCM-10178) and E.coli BL21(DE3)/pT14OSSIα-2b-4T20V22Q (HM 10712).